During the last years, methods have been introduced wherein aggregates formed between specific binding agents and bindable substances are detected by labelling said aggregates directly or indirectly with small size metal particles, particularly gold particles. Depending on the circumstances, these particles can be detected, e.g. by direct visual examination, by microscopic or spectrophotometric techniques. A description of the "immunogold staining (IGS) technique", "the sol particle immuno assay (SPIA) technique" or specific applications and improvements thereof can be found in U.S. Pat. Nos. 4,313,734, 4,446,238, 4,420,558, EP-A-0,165,633, EP-A-0,165,634, EP-A-0,158,746, EP-A-0,293,947 and in IBRO handbook series, Wiley, New York, 1983, pages 347 to 372.
Starting from a relatively unknown method for labelling cell surface antigens, metal particles have today become widely used in a variety of detection and/or quantitive determination problems. The possibility of direct visual examination of metal particles and the advantage that the signal generated is permanent and not prone to rapid degradation makes it an interesting marker for simple and rapid assays. Moreover metal markers, preferably gold markers, seem preferable over radioisotope markers due to the very low health hazard related to working with the former.
Next it was found that the signal of a metal marker such as colloidal gold can be augmented significantly by subjecting the colloidal gold markers to a so-called physical enhancement procedure. The effect of such a physical enhancement procedure is that the typical reddish optical gold signal turns into a deep-brown to black silver signal having a much higher intensity. In said procedure, the metal markers used as a label catalyze the reduction of silver ions present in the developing solution. The latter results in a specific deposition of a metallic silver layer at the metal particle site. The thus formed metallic silver particles in turn catalyze the reduction of more silver ions from the physical developing solution, creating an autocatalytic process.
Art-known physical developing solutions generally consist of a solution containing a soluble metal salt such as silver nitrate, a reducing agent such as hydroquinone, an appropriate buffer and optionally a complexant to tie up the metal ions and make them less susceptible to reduction.
Although the use of these art-known physical developers results in an augmented signal, there are a number of drawbacks associated with it. Indeed, it is well known that silver ions may form light sensitive salts such as silver bromide and silver chloride which are readily reduced to metallic silver under the influence of light, starting an autocatalytic process. This non-specific process of self-nucleation contributes to the occurence of background to a significant extent. It may be disturbingly strong under conditions of intense light, e.g. in the monitoring of test samples under a light microscope, or also when the physical development process is slow and the test sample is exposed to light for a prolonged time.
Consequently, the aim of the present invention is to provide a sensitive and practicable physical developer for use in a variety of metal based assays which is light-stable and does not give rise to undesired non-specific deposition of metal particles.
The present invention relates to a physical developer comprising an aqueous solution of silver ions, a reducing agent, a desensitizing agent and, if desired, a buffer system and one or more adjuvants.
Further there is provided a method for qualitatively and/or quantitatively determining one or more components of an aggregate formed between at least one specific binding agent and its corresponding bindable substance, which comprises labelling at least one component of said aggregate with a marker and contacting said aggregate with a physical developer whereby under influence of the marker a silver particle is formed which can be determined qualitatively, characterized in that the physical developer is a physical developer according to the present invention, comprising a solution of silver ions, a desensitizing agent and a reducing agent.
Another aspect of the present invention is to provide versatile products such as test-kits adapted for carrying out the above mentioned methods.
In the present invention the light-sensitivity of the traditional physical developers based on silver ions is counteracted by adding a desensitizing agent to the developer. Preferred desensitizing agents for use in the present invention are electron acceptors such as, 6-ethoxy-1-methyl-2-(3-nitrostyryl)quinolinium methyl sulfonate known as Pinakryptol Yellow.RTM. and the like desensitizing agents.
The reducing agents for use in the present physical developer include any agents which reduce silver ions from a physical developer in proximity of an active site. Preferably said reducing agents form stable solutions with the constituents of the improved physical developer. As a reducing agent there may particularly be mentioned, 1,2-dihydroxybenzene, 1,4-dihydroxybenzene (Hydroquinone), 4-methylaminophenolsulfate (Metol.RTM.), 4-aminophenol, 1,4-diaminobenzene, 1,2-diaminobenzene, N-(4-hydroxyphenyl)glycine, 2,4-diaminophenol, 1-phenyl-3-hydroxypyrazole (Phenidone.RTM.) or mixtures thereof. As other adjuvants of the improved physical developer there may be mentioned, buffers, preservatives, e.g., anti-oxidants or organic stabilizers, speed regulators, bactericides and the like, such as, for example, sodium sulfite, sodium bisulfite, sodium citrate and the like.
Suitable pH adjusting agents are for example, acetic acid, citric acid, sodium hydroxide or a salt of any of these or a buffer system based on tris(hydroxymethyl)aminomethane. The pH of the physical developer preferably ranges from about 5 to 9, in particular from about 6 to 8. In general the pH of the physical developer is limited to the range wherein the specific binding agent and the corresponding bindable substance are stable.
Apart from the silver ions, the reductant and desensitizing agent, the preferred physical developing solution also comprises an excess of a complexant to tie up the metal ions and to make them less susceptible for reduction. Favourable complexants for use in the present invention are described in EP-A-0,293,947 and include pyridine, aminopyridine, nicotinamide, quinoline, imidazole, histidine, benzimidazole, pyrazole, purine and the like aromatic heterocyclic ring systems.